Method and apparatus for prokaryotic and eukaryotic cell quantitation

ABSTRACT

This invention describes methods and kits for detecting and quantifying viable cells in a sample using fluorescent dyes that can be internalized predominantly by viable cells and have fluorescent properties measurably altered when bound to target components. These methods and kits provide a rapid and cost-effective means of detecting potential biological threats in the field.

CROSS-REFERENCES TO RELATED APPLICATIONS

[0001] This application is a continuation-in-part of U.S. patentapplication Ser. No. 09/696,710, filed on Oct. 24, 2000, now pending,which claims priority to U.S. Provisional Application No. 60/163,738,filed on Oct. 25, 1999, all of which are incorporated herein byreference in their entirety.

TECHNICAL FIELD

[0002] The present invention relates generally to methods for the rapiddetection of viable cells involving staining viable cells in a samplewith a fluorescent dye and measuring the fluorescence. The invention ismore particularly related to methods and kits for the rapididentification of microbes in an environmental sample.

BACKGROUND OF THE INVENTION

[0003] Biowarfare and bioterrorism include the intentional or thealleged use of viruses, bacteria, fungi and toxins to produce death ordisease in humans, animals or plants. While biowarfare poses substantialhealth risks, bioterrorism also possesses the ability to severelydisrupt individuals' lives, as well as societies and economies.Unfortunately, hoaxes and false alarms similarly disrupt lives and work.Individuals affected by both credible and false biowarfare threats areoften forced to wait long time periods while officials determine if acredible biological threat exists, even though only one or two out ofseveral hundred responses may result in a credible threat. Clearly,methods of rapidly determining whether a credible biological threatexists are vitally important in preventing both disease and societal andeconomic disruption.

[0004] A variety of methods are currently employed for the detection ofmicrobial biowarfare agents, such as bacteria and yeast. These methodsinclude, but are not limited to, conventional cell viability assays suchas the standard plate count, dye reduction and exclusion methods,electrometric techniques, microscopy, flow cytometry, bioluminescence,colorimetry, and turbidity. However, such methods are generally notuseful for rapid detection, as required for field testing and rapiddetermination of the existence of a potential biological threat. Thesemethods typically require highly trained personnel and expensiveinstrumentation. In addition, many of these assays require substantiallypure populations of cells or long incubation periods.

[0005] Additional methods specifically designed to detect biologicalthreats include, for example, immunodiagnostic assays, DNA assays usingreal-time polymerase chain reaction (PCR), mass spectrometry, enhancedbioluminescence assays, protein nanoarrays, volatile organic compoundanalysis, and multi photon detection. Again, most of these assaysrequire expensive instrumentation and highly-trained personnel, andcannot be rapidly performed in the field.

[0006] Several handheld kits for the detection of biowarfare agents havebeen described. Sensitive Membrane Antigen Rapid Test (SMART™) Ticketsare currently sold commercially for the rapid detection of Bacillusanthracis spores, V. cholerae serotype 01, Yersina pestis, and Botulismtoxin. SMART™ Tickets employ a calorimetric immunoassay designed for thedirect presumptive recognition or detection of a presumed biowarfareagent(s). However, the analytical sensitivity of these assays is limitedby the current technology, and data provided by manufacturers indicatethat a minimum of 10,000 spores is required to generate a positivesignal. This number of spores would require a relatively heavycontamination of the area (sample) being tested.

[0007] Bio Threat Alert™ test strips are currently marketed for thedetection of Bacillus anthracis, ricin toxin, Botulinum Toxin ,Staphococcal Enterotoxin B, Yersina pestis, and Francisella tularensis.The Bio Threat Alert™ test strip employs agent-specific antibodies topositively identify the potential threat. Screening results are producedin approximately 15 minutes and may be read visually or using theGuardian Bio Threat Alert™ Test Strip Reader.

[0008] Although SMART™ Tickets and Bio Threat Alert test strips aredesigned for rapid field detection of biowarfare agents, theirusefulness appears limited by low sensitivity of detection and theirability to detect a very limited number of microbial biowarfare agents.Further, such antibody-dependent tests are susceptible to thepossibility of designed or naturally occurring biological mechanismsthat would result in an escape from immune detection. For example,several standard dual or multidye fluorescence filter set are known andavailable from microscope and filter manufacturers including Zeiss,Leica, Chroma Technologies, Nikon and the like.

[0009] The present invention for the detection and quantitation of cellsis designed to overcome problems that have been identified within thefield. The present technology circumvents the need for trainingpersonnel in how to plate, grow and count viable cells from colonies onagar plates and eliminates nearly all training and maintenance costsassociated with most other microbial biowarfare agent detection andquantification methods. In addition, the invention substantiallydecreases the time needed for biowarfare agent detection, permittingmore accurate detection in less than twenty minutes, while stillallowing for the detection and quantification of any live cell presentin the test sample. Finally, the technology offers substantial costsavings over most existing methods of microbial biowarfare agentdetection and quantitation.

BRIEF SUMMARY OF THE INVENTION

[0010] Briefly, the present invention describes methods and kits fordetecting and quantifying viable cells in a sample using fluorescentdyes that can be actively internalized by viable cells and havefluorescence properties measurably altered when bound to targetcomponents.

[0011] In one embodiment of the present invention, a method forquantifying viable cells in a sample is disclosed, comprising thefollowing steps: (1) contacting a sample with a fluorescent dye, whereinthe dye is actively or passively internalized by the cells and hasfluorescence properties that are measurably altered when bound to targetcomponents; (2) detecting total fluorescence of the sample; and (3)comparing the fluorescence produced by the sample to the fluorescenceproduced by a control substance. Within certain embodiments, (1) thecells in the sample are bacteria, spores, and yeast and other fungi; (2)the fluorescent dye binds to DNA of the viable cells; (3) the sample istreated with DNase before it is mixed with the fluorescent dye; (4) thesample is treated with an agent that affects a cell membrane property ofthe cells (e.g., a detergent) prior to, subsequently or concurrentlywith the fluorescent dye; or (5) the fluorescent dye is acridine orange,Hoechst 33258, PicoGreen™, SYTO® 16, SYBR® Green I, Texas Red®, RedmondRed™, Bodipy® Dyes, or Oregon Green™.

[0012] In certain embodiments, the cells in the sample are Bacillusanthracis, Bacillus cereus, Clostridium botulinum, Yersinia pestis,Yersinia enterocolitica, Francisella tularensis, Brucella species,Clostridium perfringens, Burkholderia mallei, Burkholderia pseudomallei,Staphylococcus species, Tuberculosis species, Escherichia coli, Group AStreptococcus, Group B streptococcus, Streptococcus pneumoniae,Helicobacter pylori, Francisella tularensis, Salmonella enteritidis,Mycoplasma hominis, Mycoplasma orale, Mycoplasma salivarium, Mycoplasmafermentans, Mycoplasma pneumoniae, Mycobacterium bovis, Mycobacteriumtuberculosis, Mycobacterium avium, Mycobacterium leprae, Rickettsiarickettsii, Rickettsia akari, Rickettsia prowazekii, Rickettsia canada,Coxiella burnetti, Aspergillus varieties, Mucor pusillus, Rhizopusnigricans, Candida albicans, C. parapsilosis, C. tropicalis, C.pseudotropicalis, Torulopsis glabrata, Aspergillus niger, Candidadubliniensis, Blastomyces dermatitidis, Coccidioides immitis,Histoplasma capsulatum, Aspergillus species, Candida species,Cryptococcus neoformans, Bacillus subtilis, Bacillus subtilus niger,Bacillus thuringiensis, all Bacillus sp. and/or Sporothrix schenckii.

[0013] The present invention also discloses kits for detecting orquantifying viable cells. One such kit comprises a cell suspensionsolution, a fluorescent dye that can be actively internalized by viablecells, and instruction for detecting dye binding to cellular componentsof viable cells. The cell suspension solution may include a DNase or anagent that affects cell membrane property, such as a detergent. Incertain embodiments, the fluorescent dye may be acridine orange, Hoechst33258, PicoGreen™, SYTO® 16, SYBR® Green I, Texas Red®, Redmond Red™,Bodipy® Dyes, or Oregon Green™. Such fluorescent dyes are readilyavailable from commercial sources including Molecular Probes (Eugene,Oreg.), Sigma Chemical (St Louis, Mo.), Amersham (Arlington Heights,Ill.), Callbiochem-Novabiochem (La Jolla, Calif.), Synthetic Genetics(San Diego, Calif.), and the like.

[0014] In another embodiment, a kit comprises a first containercontaining a first solution, means for placement of a sample containingan unknown number of viable cells into a solution containing afluorescent dye that can be internalized predominately by the viablecells and binds selectively to double-stranded DNA or other specificcellular components, whereupon its fluorescence is altered to ameasurable degree, and means for illuminating the mixture of firstsolution with said sample with excitation light and measuringfluorescence emitted by said mixture, thereby detecting the presence ofviable cells in said sample.

[0015] In yet another embodiment, a kit comprises a first containercontaining a first solution, means for placement of a sample containingan unknown number of viable cells into a first solution, means forconcentrating the solids and cells from the mixture of said firstsolution with said sample and retaining said solids from the remainderof said mixture, a second solution containing a fluorescent dye that canbe internalized predominately by the viable cells and binds selectivelyto double-stranded DNA or other specific cellular components, whereuponits fluorescence is altered to a measurable degree, means for mixingsaid second solution with said solids to form a second mixture, andmeans for illuminating the mixture of said second solution with saidsolids with excitation light and measuring fluorescence emitted by saidmixture, thereby detecting viable cells in said sample.

[0016] Upon binding, the fluorescence properties of the fluorescent dyeare altered to a measurable degree. The first solution may contain aDNase or an agent that affects a bacterial cell membrane property, suchas a detergent. Within certain embodiments, the fluorescent dye may beacridine orange, Hoechst 33258, PicoGreen™, SYTO® 16, SYBR® Green I,Texas Red®, Redmond Red™, Bodipy® Dyes, or Oregon Green™.

BRIEF DESCRIPTION OF DRAWINGS

[0017]FIG. 1 is a correlation of relative intensities of the fluorescentemission (TBAK readings) to the standard plate counts (colony formingunits/ml) of Mesophilic Bacteria.

[0018]FIG. 2 is a chart that correlates relative fluorescence readingswith colony forming units/ml of Mesophilic Bacteria.

[0019]FIG. 3 is a graph depicting the relationship of fluorescence tospore concentration using a nucleic acid binding dye such as SYTO® 16.

[0020]FIG. 4 is a bar graph depicting relative light units of commonsubstances and comparison of the same to several microbes.

DETAILED DESCRIPTION OF THE INVENTION

[0021] As noted above, the subject invention is concerned with methodsand kits for detecting and quantitating viable cells in a sample. Theinvention is applicable to the detection and quantitation of all typesof viable cells, including eukaryotes, such as plant and animal cells(e.g., mammalian cells, particularly human cells), and prokaryotes,particularly bacteria (including both Gram-positive and Gram-negativebacteria), bacterial spores, yeast, and other fungi. Viable cells arecells that have intact cell membranes and are actually or potentiallymetabolically active. As used herein, spores and cysts are examples ofviable cells.

[0022] In certain embodiments, the methods and kits of the invention areused to detect or quantitate biowarfare agents, including infectiousagents such as bacteria and fungi, for example. Examples of bacteria andspores that may be detected according to the present invention include,but are not limited to, Bacillus anthracis, Bacillus cereus, Clostridiumbotulinum, Yersinia pestis, Yersinia enterocolitica, Francisellatularensis, Brucella species, Clostridium perfringens, Burkholderiamallei, Burkholderia pseudomallei, Staphylococcus species, Tuberculosisspecies, Escherichia coli, Group A Streptococcus, Group B streptococcus,Streptococcus pneumoniae, Helicobacter pylori, Francisella tularensis,Salmonella enteritidis, Mycoplasma hominis, Mycoplasma orale, Mycoplasmasalivarium, Mycoplasma fermentans, Mycoplasma pneumoniae, Mycobacteriumbovis, Mycobacterium tuberculosis, Mycobacterium avium, Mycobacteriumleprae, Rickettsia rickettsii, Rickettsia akari, Rickettsia prowazekii,Rickettsia canada, and Coxiella burnetti. Examples of yeast and otherfungi that may be detected according to the invention include, but arenot limited to, Aspergillus species (e.g. Aspergillus niger), Mucorpusillus, Rhizopus nigricans, Candida species (e.g. Candida albicans,Candida dubliniensis, C. parapsilosis, C. tropicalis, and C.pseudotropicalis), Torulopsis glabrata, Blastomyces dermatitidis,Coccidioides immitis, Histoplasma capsulatum, Cryptococcus neoformans,and Sporothrix schenckii.

[0023] Methods and kits of the invention may be used to detect and/orquantitate infectious agents associated with diseases, including, butnot limited to, cutaneous anthrax, inhalation anthrax, gastrointestinalanthrax, nosocomical Group A streptococcal infections, Group Bstreptococcal disease, meningococcal disease, blastomycocis,streptococcus pneumonia, botulism, Brainerd Diarrhea, brucellosis,pneumonic plague, candidiasis (including oropharyngeal, invasive, andgenital), drug-resistant Streptococcus pneumoniae disease, E. coliinfections, Glanders, Hansen's disease (Leprosy), cholera, tularemia,histoplasmosis, legionellosis, leptospirosis, listeriosis, meliodosis,mycobacterium avium complex, mycoplasma pneumonia, tuberculosis, pepticulcer disease, nocardiosis, chlamydia pneumonia, psittacosis,salmonellosis, shigellosis, sporotrichosis, strep throat, toxic shocksyndrome, trachoma, traveler's diarrhea, typhoid fever, ulcer disease,and waterborne disease.

[0024] As those of ordinary skill in the art readily appreciate, thesample can be from any source. According to the present invention, thesample is typically an unknown substance, such as a powder or liquid,for example. Samples may be found outdoors or indoors. In oneembodiment, a sample is an unknown powder found in or on an envelope,package, or other item of mail. In another embodiment, the sample is abodily fluid such as blood, urine, spinal fluid, or other similarfluids. Alternatively, the sample is a fermentation medium such as froma biological reactor or food fermentation process such as brewing. Thesample may also be food products such as milk, yogurt, cheese, meats,beverages and other foods. Other sources for samples include water,small amounts of solids, or liquid growth medium.

[0025] In one embodiment, viable cells in a sample are detected orquantified by comparing the fluorescence produced by the sample to thefluorescence produced by a control substance. Useful control substancesare substances that do not contain live cells, including common powderssuch as, but not limited to, sugar, salt, baking soda, and powder coffeecreamer. Fluorescence produced by the sample that is significantlygreater than the fluorescence produced by the control substanceindicates the presence of live cells in the sample. Significantdifferences should be at least two-fold and preferably 10-fold orgreater.

[0026] Alternatively, or in addition, viable cells, and a viable viraland/or non-viral infectious agent, in a sample are detected by comparingthe fluorescence produced by the sample to the fluorescence produced bya positive control substance. In some embodiments a means for enrichingviable cells from viable viral or non-viral biowarfare agent iscontemplated. A positive control substance may comprise any cell ormolecule capable of binding the fluorescent dye to produce a measurablechange in fluorescence. Suitable positive control substances mayinclude, for example, DNA, bacteria, and yeast.

[0027] In another embodiment, viable cells in a sample are detected bycomparing the fluorescence produced by the sample to standardmeasurements. Standards generally provide typical fluorescencemeasurements associated with different numbers and/or types of cells orDNA, for example. Standards, including those produced by controlsubstances, may be determined in advance of testing of a sample in thefield, or they may be prepared at approximately the same time or aftertesting a sample. Standard measurements may also be provided by amanufacturer or test laboratory.

[0028] The present invention requires that fluorescent dyes be activelyinternalized by viable cells in a sample and bind to their targetcellular components. In addition, the invention requires that uponbinding, the fluorescence properties of the dyes be altered to ameasurable degree. The term “actively internalized” refers tofluorescent dyes that penetrate predominantly viable cells through aprocess other than passive diffusion across cell membranes. It includes,but is not limited to, internalization of fluorescent dyes throughreceptors on cell surfaces or through channels in cell membranes. Asused herein, the term “actively internalized” may be interchangeablyreferred to as “internalized predominantly.”

[0029] The target cellular components to which fluorescent dyes bind maybe nucleic acids, actin, tubulin, nucleotide-binding proteins,ion-transport proteins, mitochondria, cytoplasmic components, andmembrane components. Fluorescent dyes that fluoresce when metabolized bythe cells in a sample, such as dequalinium aceteate, fluoresceindiacetate, or other similar compounds, are also included in theinvention.

[0030] Examples of fluorescent dyes that bind nucleic acids and suitablefor the present invention include, but are not limited to, acridineorange (U.S. Pat. No. 4,190,328), calcein-AM (U.S. Pat. No. 5,314,805),DAPI, Hoechst 33342, Hoechst 33258, PicoGreen™, SYTO® 16, SYBR® Green I,Texas Red®, Redmond Red™, Bodipy® Dyes, Oregon Green™, ethidium bromide,propidium iodide, and the like. When such fluorescent dyes used toquantify viable cells in a sample, it is preferable there are no morethan small differences in the nucleic acid content among the individualcells in the sample. The quantification method works accurately forsamples containing predominantly a single species of bacteria or otherspecific cell types under normal growth conditions, such as in cheesestarter cultures. However, variation in DNA content of microbes inaxenic or microbial consortia cultures affects quantitative measurementsless than the variation affects standard and other methods.

[0031] The present invention's detection and quantitative accuracy mayalso be affected by such factors as effective penetration of viablecells by the dye (which can be readily calculated for any dye), a lowlevel of background fluorescence relative to the amount of fluorescenceof dye bound specifically to DNA or other cellular components, lowlevels of exogenous DNA in the sample, and so on.

[0032] PicoGreen™ and SYTO® 16 are dyes particularly useful to thepresent invention and commercially available from Molecular Probes,Eugene, Oreg. PicoGreen™ is a cyanine dye with high molar absorptivityand very low intrinsic fluorescence. PicoGreen™ is specific for doublestrand DNA and produces large fluorescence enhancements (over 1000-fold)upon binding to DNA. Haugland, Richard P., Handbook of FluorescentProbes and Research Chemicals, Molecular Probes, Eugene, Oreg., (1996).However, until the present invention it was not known to be taken uppredominantly by viable cells. SYTO® 16 is a cell-permeant nucleic acidstain capable of staining most live and permeabilized bacteria, as wellas eukaryotic cells. In addition, SYTO® 16 has proven particularlyuseful for detecting and/or otherwise distinguishing live from deadcells. Haugland, Richard P., supra.

[0033] Dyes are typically prepared by dissolving the dye in an aqueoussolvent such as water, a buffer solution, or a water-miscible organicsolvent such as dimethylsulfoxide (DMSO), dimethylformamide (DMF),methanol or ethanol. In certain embodiments, it is dissolved at aconcentration of greater than about 100-times that used for stainingsamples. Preferably, the staining solution imposes no or minimalperturbation of cell morphology or physiology.

[0034] Before contacting fluorescent dyes, the sample or a fraction ofthe sample may first be rinsed in a buffer solution and centrifuged,filtered, or otherwise concentrated. The initial rinse solutions can bemade in aqueous or other polar or semi polar solvents containing varioussalts between 0.05M and 1.0 M with a final pH of between 3.5 and 11.0 attemperatures between −20 degrees C. and 80 degrees C. The solutions mayalso contain reagents that maximize integrity of viable cells whilereleasing non-cellular materials into the solutions. Such reagents mayinclude, by way of example but not in limitation, phosphates, neutral,anionic and cationic detergent (e.g., Tween® NP and Triton® seriesdetergents, SDS, and cetyl-trimethyl ammonium BR, chaotropic salts(e.g., bile salts), organic acids (e.g. citrate), and lipids. Inaddition, when a fluorescent dye specific for DNA is used, the samplemay be treated with a DNase to remove exogenous DNA before it iscontacted with the dye.

[0035] As noted above, the fluorescent dyes need to be internalized bythe cells to be detected and/or enumerated. In the preferred inventivemethod, an excess of the dye is used in order to saturate the cellularDNA in the sample. This can be determined by adding an amount known tobe enough, or else by adding more and more until fluorescence ceases toincrease.

[0036] In certain embodiments, multiple dyes may be used in order todetect more than one type of potential bioweapon. Means for multibanddetections systems are well known in the art, for example, by way ofexample but not limitation, a multiwavelength fluorometer.

[0037] In certain embodiments, after contamination is washed from thecells, an agent that affects cell membrane properties may be used totreat the viable cells to speed the internalization process. Such agentsinclude, but are not limited to, detergent-like compounds, surfactants,or other compounds that affect membrane polarity, fluidity,permeability, potential gradient or other membrane properties.Alternatively, DNA from the viable cells may be first extracted and thencontacted with a fluorescent dye specific for DNA.

[0038] In the case that a sample is highly translucent, fluorescent dyesmay be directly added to the sample and the relative fluorescence isthen directly measured.

[0039] After being contacted with fluorescent dyes, the sample isexcited by a light source capable of producing light at or near thewavelength of maximum absorption of the fluorescent complex, such alaser, an arc lamp, an ultraviolet or visible wavelength emission lamp.Any apparatus or device that can measure the total fluorescence of asample can be used in this invention including flow cytometers.Typically, a standard fluorometer is used because of its relative lowcost and field accessibility. The instrumentation for fluorescencedetection may be designed for microscopic, surface, internal, solution,and non-suspension sample formats. The total fluorescence measured isthen used to calculate the number of viable cells in the sample based ona predetermined value establishing a statistically relevant correlationbetween total fluorescence and the number of viable cells measured usingany prior art method (e.g., standard plate count). The fluorescence maybe measured at three or more intervals as the dye is entering the cells,so that the final fluorescence can be predicted in advance ofsaturation, thereby the time required to obtain a cell count isshortened. One of ordinary skill in the art would readily understandthat any light source or measurement technique maybe used.

[0040] The invention also discloses a method of quantifying the ratio ofviable cells to dead cells in a sample. In this embodiment, a samplecontaining both viable and dead cells is first saturated with aninternalizing fluorescent dye. This dye does not need to be activelyinternalized by viable cells. It may diffuse into both viable and deadcells. After saturation, a second dye having emission wavelengthsoverlapping with those of the first dye is added to the mixture of thesample and the first dye. The second dye is capable of beinginternalized predominately by the viable cells in the sample. Theinternalization of the second dye quenches the fluorescence emitted bythe first dye. The quenching effect of the second dye on the first dyecan be used to calculate the ratio of viable cells to dead cells in thesample.

[0041] The present invention also discloses kits and apparatuses fordetecting and quantifying viable cells. One such kit comprises a cellsuspension solution, a fluorescent dye, and instruction for detectingthe binding of the dye to cellular components of the viable cells andcorrelating the binding to cell number or colony forming units. Thesuspension solutions may be aqueous or other polar or semi-polarsolvents containing various salts between 0.05M and 1.0 M with a finalpH of between 3.5 and 11.0 at temperatures between −20 degrees C. and 80degrees C. The solutions may also contain reagents that maximizeintegrity of viable cells while releasing non-cellular materials intothe solutions as described above, a DNase, or an agent that affects cellmembrane properties, such as a detergent. The fluorescent dye in the kitis capable of being actively internalized by the viable cells, bindingto cellular components and altering its emission of fluorescence uponthe binding to a measurable degree. The instruction contains necessaryinformation of how to use the kit to detect or quantify viable cells ina sample, such as how to detect the fluorescence emitted from the dyeand how to correlate the fluorescence measured with cell number orcolony forming units.

[0042] In certain embodiments, kits may also include tools or devicesuseful for isolating a sample and/or placing a sample into a containeror solution. For liquid samples, such tools or devices typically includepipettes and the like. For solid or powder samples, such tools ordevices include scoops, swabs, cups, tubes, and the like, for example. Apipette may also be used for solid or powder samples. One of ordinaryskill would recognize that a wide variety of tools may be used for thispurpose.

[0043] In another aspect, a kit for quantifying live bacteria in asample may include any one or combinations of the following: a cellsuspension solution, means for placement of a sample into the solution,means for mixing the cell suspension solution with the sample, means forconcentrating the cells, a fluorescent dye solution, means for mixingthe dye solution with concentrated cells, and means for illuminating theresulting mixture with excitation light and measuring fluorescenceemitted. The cell suspension solution may be the same as in the otherkit described above. Any means for mixing solution with a sample knownin the art (e.g., pipets, vortex) can be included in this kit. Any meansfor concentrating cells from solutions known in the art (e.g.,centrifugation, filtration) may also be included in the kit. Thefluorescent dye in the kit is capable of being actively internalized bythe viable cells, binding to cellular components and altering itsemission of fluorescence upon the binding to a measurable degree. Anymeans for illuminating a fluorescent solution with excitation light andmeasuring fluorescence emitted known in the art (e.g., flow cytometers,fluorometer) may be included in the kit.

[0044] Refinements of the above-described kits may include other dyesthat bind uniquely to other cellular components of the sample, means forspectral and time-resolved analysis of the emitted fluorescent light,and means for separating the cell components to which the fluorescentdye binds from the sample before contacting with the fluorescent dye.

[0045] Depending on the properties of the fluorescent dye, the disclosedmethods and kits may also be used in various assays other than viablecell identification and/or quantification. For instance, the use offluorescent dyes that are capable of differentiating two kinds of cells(e.g. different bacteria or bacteria from yeast) enables the measurementof the ratio between the two kinds. The use of a fluorescent dyespecific for a particular enzymatic activity may also be used to detect,identify, or quantify cells using the particular enzymatic activity. Forexample, fluorescein diacetate may be used to enumerate cells withactive esterase. Such a fluorescent dye can be further used to enumeratecells in which a particular enzymatic activity is induced by certainchemicals (e.g., naphthalene and dequalinium acetate for the inductionof esterase activity) or treatments (e.g., heat). In addition,fluorescent dyes that differentiate prokaryotes and eukaryotes based ontheir differences in cell membrane proteins, the presence or absence ofcertain organelles, and metabolism may be used to quantify prokaryoticcells, eukaryotic cells, or the ratio between the two types of cells.Furthermore, fluorescent dyes of which intensity is enhanced bycontacting nucleated or enucleated cells in a sample that are known tobe dead or not actively metabolizing in a sample may be used toenumerate such cells.

[0046] In other embodiments, fluorescent dyes that quench upon bindingto specific cellular components after being predominantly internalizedby viable cells may also be used to detect, identify, or quantify viablecells. In yet other embodiments, dyes or other substances that changethe NMR signature of cells or any other bulk-detectable property of thecells may also be used for enumerating cells.

[0047] The present invention provides methods, kits, and apparatuses forsimple dye associated identification or quantification that allows oneto rapidly and inexpensively determine the presence and/or number ofviable cells in a particular sample. One of ordinary skill in the artwill readily appreciate that alternatives to the steps herein describedfor detecting or quantifying cells may be used and are encompassedherein. Accordingly, all alternatives will use a kit or method wherein adye is utilized to stain cells and a detection method. Key aspects ofthe present invention include its rapid detection of viable cells andsubstantial cost savings as compared to previous systems.

[0048] All patents, patent applications and references cited herein areincorporated herein in their entirety. Accordingly, incorporated hereinby reference are U.S. Pat. Nos. 5,437,980; 5,563,070; 5,582,984;5,658,751; 5,436,134; Catt, S. L., Sakkas, D., Bizzaro, D., Bianchi, P.G., Maxwell, W. M. and Evans, G.; (1997) Molecular and HumanReproduction 3:821-825; Ferguson, L. R., and Denny, W. A.; (1995)Mutation Research 329:19-27; and Latt, S. A. and Wohlleb, J. D.; (1975)Chromosoma 52:297-316.

[0049] From the foregoing it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention. Accordingly, the invention is notlimited except as by the appended claims.

[0050] The following examples are offered by way of illustration, andnot by way of limitation. All of the references, including patents,patent applications, and journal references are hereby incorporated byreference in their entirety.

EXAMPLES Example I Quantitation of Bacterial Cell Number

[0051] This example illustrates a method of enumerating live bacterialcells.

[0052] Solutions

[0053] Solution A. This is a wash/isolation solution that prepares thecells to take up the dye and permits removal of any interferingexogenous substances. All chemicals are reagent grade and can beobtained from common suppliers in the trade.

Ingredients and Preparation and Storage of Solution A (Ten TimesConcentrate)

[0054] Ingredients: NaCl 80.0 grams KCl  2.0 grams Na₂HPO₄ 14.4 gramsKH₂PO₄  2.4 grams NaOH sufficient to reach pH 7.4 Pure water sufficientfor 1 liter

[0055] Preparation:

[0056] The above chemicals are dissolved in 850 ml of pure water at roomtemperature. The pH is adjusted to 7.4 with 1M NaOH. The volume isadjusted to 1 liter with pure water. The solution is autoclaved and maybe stored at room temperature for up to one year.

[0057] Ingredients Preparation and Storage of Stock Solution of SodiumDodecyl Sulfate

[0058] Ingredients:

[0059] Sodium dodecyl sulfate (SDS) . . . 2.0 grams

[0060] Preparation:

[0061] Dissolve 2.0 grams of SDS in enough pure water to make a finalvolume of 100 ml.

[0062] Storage:

[0063] The solution may be stored for up to one year at roomtemperature.

[0064] Ingredients, Preparation and Storage of Working Solution A

[0065] Ingredients: Solution A (Ten Times Concentrate) 100 ml StockSolution of SDS  0.5 ml Pure water 900 ml

[0066] Preparation:

[0067] Add the above solutions to a 1 liter container. Solution A isused in the invention.

[0068] Storage:

[0069] Solution A is autoclaved and then stored closed at roomtemperature.

[0070] Solution B. This solution contains a dye that stains viablecells. The formulation may allow unique stability and rapid uptake ofthe dye by the viable cells. All chemicals are reagent grade and can beobtained from common suppliers in the trade such as Sigma Chemical Co.,St. Louis, Mo.

[0071] Ingredients and Preparation and Storage of Solution B.

[0072] Ingredients: Hoechst 33258 100 mg Sterile pure water  10 mlPropylparaben  0.1 ml (approximately-- this amount is not critical)

[0073] Preparation of Solution B One Hundred Times Concentrate (anexample using Hoechst 33258):

[0074] Solution B is made by dissolving Hoechst 33258 or similar dye insterile pure water to a final concentration of 10 ug/ml. An antioxidantsuch as propyl gallate or propylparaben is added to increase shelf lifeof the solution.

[0075] Storage:

[0076] This solution is stored in the dark at 4 degrees C. It is stablefor at least 6 months.

[0077] Dilution of Solution B

[0078] Ingredients: Solution B (One Hundred Times Concentrate) 0.1 mlSterile, pure water 9.9 ml

[0079] Dilution:

[0080] Solution B (One Hundred Times Concentrate) is diluted {fraction(1/100)} in sterile, pure water. For example, 100 ml of Solution B oneHundred Times Concentrate is dissolved into 9.90 ml of sterile, purewater for a final concentration of 0.1 mg/ml.

[0081] Reaction Solution Preparation for SYTO® 16 Dye and SporeMeasurement

[0082] Remove frozen sample of 1 mM SYTO® 16 dye from the freezer andplace on desktop to thaw (10-20 min).

[0083] Carefully pipet 1 ml of DMSO into a clean, autoclavedpolypropylene tube.

[0084] Use a calibrated pipet to remove 50 μl of thawed SYTO® 16 dyefrom the sample vial, and add it to the tube containing 1 ml DMSO. Thiswill yield a 50 μM final concentration for the working ReactionSolution.

[0085] Cap the polypropylene tube, and immediately wrap with aluminumfoil, covering the sides and top.

[0086] Vortex the tube for a few seconds to ensure complete mixture ofthe chemicals.

[0087] Cell Prep Solution Preparation (TNE buffer)

[0088] *Recipe calls for: 10 mM Tris, 1 mM EDTA, 50 mM NaCl-titrate topH 7.4

[0089] Measure out approximately 985 ml of deionized/distilled water ina large graduated cylinder and pour into a large Erlenmeyer flask. Setaside

[0090] In a weigh boat, carefully weigh out 1.576 g of Trizma HCl. Addto flask.

[0091] Repeat measurement, weighing out 2.922 g of NaCl. Add to flask.

[0092] Measure out 282 mg of EDTA powder OR use 11.76 ml of prepared0.085M EDTA solution. Add to flask.

[0093] Agitate flask to ensure that all chemicals mix togetherthoroughly.

[0094] Using NaOH and a Pasteur pipet, titrate cell prep solution to apH of 7.4. If necessary, top off solution with deionized/distilled waterto a final volume of 1000 ml.

[0095] Autoclave and sterile filter solution.

[0096] Sample Collection and Fluorescence Measurement

[0097] One particularly useful fluorometer is the GP320 hand-heldfluorometer by Turner Designs in Sunnyvale, Calif. Any fluorometer withfilters appropriate for the dye used is useful. The wavelengths used forthe detection of spores using SYTO® 16 in the Prime Alert™ method are:

[0098] 475 nm excitation and 515 mn emission.

[0099] Calibrate the GP320 reader with the solid “B” calibrationstandard, making sure that the GP320 is in B mode. For calibrationinstructions, see page 8 of the Prime Alert Instruction Manual. Tochange the mode on the GP320, push the A/B button until “B” appears inthe data screen.

[0100] Use the pre-filled Prime Alert dropper bottle to fill an emptytest vial with 8 drops of Prime Alert cell prep solution. Set test vialaside.

[0101] Using the Prime Alert sampling device, collect one scoop ofsample powder. Use the spatula to gently pack and level off the scoop,and to eliminate excess powder from the scoop edges and bottom.

[0102] Remove the screw-top lid and empty the scoop into the dropperbottle. To ensure that the entire sample has been ejected from thescoop, tap the scoop against the walls of the dropper bottle. Ifpossible, avoid submerging the scoop in the Prime Alert cell prepsolution.

[0103] Replace the dropper bottle screw-top lid, and tighten. With thecap secure on the end of the spout, vigorously shake the dropper bottlefor several seconds. Remove the cap from the end of the spout and add 4drops of the sample solution into the previously filled test vial.

[0104] Using the transfer pipet, draw the 50 μM Prime Alert ReactionSolution into the pipet, and then expel the full volume into the testvial containing the sample solution. Cap and shake the test vial, thenplace it in the GP320. Press READ.

[0105] Methods

[0106] The procedure given below discloses an example of a method forquantitating bacteria present in a liquid suspension using theinvention.

[0107] 1. Calibration and Blanking of the Fluorometer

[0108] A simple table-top fluorometer such as Turner Designs model 360is used which is fitted with a set of filters, 365 nm excitation lightwavelength and 460 nm emission light wavelength. A calibration solutionof known fluorescence intensity is employed to calibrate the instrumentto a reading of zero. A blank solution such as Solution A is used to setthe instrument to a reading of zero. All procedures are carried out atroom temperature.

[0109] Blanking Procedure:

[0110] a) place blank standard solution in reader (fluorometer) cuvette

[0111] b) close lid of fluorometer and push “blank” on keypad

[0112] c) adjust the fluorometer to read 5000 relative fluorescent units

[0113] Calibration Procedure:

[0114] a) place unique “calibration solution” in cuvette

[0115] b) close lid of fluorometer and push “calibration” on keypad

[0116] c) adjust the fluorometer to read 5000 relative fluorescent units

[0117] 2. Method for counting bacteria

[0118] a) Add 200 microliters of Solution A to the cuvette containingbacterial cells.

[0119] b) Add a 5 microliter volume of bacterial cells to Solution A inthe cuvette.

[0120] c) Centrifuge at 2000 g for 30 seconds.

[0121] d) Remove the supernatant solution by shaking or pipetting toremove it while retaining the cells.

[0122] e) Add 200 microliters of Solution A to the cuvette containingbacteria and vortex to suspend the cells.

[0123] f) Centrifuge at 2000 g for 30 seconds.

[0124] g) Remove the supernatant solution by shaking or pipetting, whileretaining the cells.

[0125] h) Add 100 microliters of Solution B, vortex to suspend the cellsand incubate for 10 min at room temperature.

[0126] i) Place cuvette in fluorometer, close the lid and record therelative fluorescence value.

[0127] j) Correlate the relative fluorescence reading with colonyforming units/ml.

[0128] Data

[0129]FIG. 2 is an example of a correlation of relative intensities offluorescence emission (TBAK readings) to the standard plate counts(colony forming units/ml). FIG. 3 is another example of correlation ofrelative fluorescence readings with colony forming units/ml in a chartform. Both figures indicate that the intensity of the fluorescenceemission is directly proportional to the cell numbers as measured by thestandard plate count method.

1. A method for detecting the presence of viable cells in a sample,comprising: a) contacting a sample with a fluorescent dye that binds totarget components of a viable cell, wherein said dye is internalizedpredominately by viable cells and has fluorescence properties that aremeasurably altered when bound to target components; b) detecting totalfluorescence of said sample; and c) comparing the fluorescence detectedin step (b) to the fluorescence produced by a control substance, therebydetecting viable cells.
 2. The method of claim 1, further comprisingcorrelating the fluorescence detected in step (b) to the number ofviable cells in the sample.
 3. The method of claims 1 or 2 wherein thecells in the sample are selected from the group consisting of: bacteria,spores, yeast, DNA containing viruses, and fungi.
 4. The method of claim3 wherein the bacteria are selected from the group consisting of:Bacillus anthracis, Bacillus cereus, Clostridium botulinum, Yersiniapestis, Yersinia enterocolitica, Francisella tularensis, Brucellaspecies, Clostridium perfringens, Burkholderia mallei, Burkholderiapseudomallei, Staphylococcus species, Tuberculosis species, Escherichiacoli, Group A Streptococcus, Group B streptococcus, Streptococcuspneumoniae, Helicobacter pylori, Francisella tularensis, Salmonellaenteritidis, Mycoplasma hominis, Mycoplasma orale, Mycoplasmasalivarium, Mycoplasma fermentans, Mycoplasma pneumoniae, Mycobacteriumbovis, Mycobacterium tuberculosis, Mycobacterium avium, Mycobacteriumleprae, Rickettsia rickettsii, Rickettsia akari, Rickettsia prowazekii,Rickettsia canada, Bacillus subtilis, Bacillus subtilus niger, Bacillusthuringiensis and Coxiella burnetti.
 5. The method of claim 3 whereinthe cells are Bacillus anthracis.
 6. The method of claim 3 wherein thespores are Bacillus anthracis, Bacillus cereus, Bacillus subtilis,Bacillus subtilus niger, and Bacillus thuringiensis.
 7. The method ofclaim 3 wherein the yeast are selected from the group consisting of:Aspergillus varieties, Mucor pusillus, Rhizopus nigricans, Candidaalbicans, C. parapsilosis, C. tropicalis, C. pseudotropicalis,Torulopsis glabrata, Aspergillus niger, and Candida dubliniensis.
 8. Themethod of claim 3 wherein the fungus is selected from the groupconsisting of: Blastomyces dermatitidis, Coccidioides immitis,Histoplasma capsulatum, Aspergillus species, Candida species,Cryptococcus neoformans, and Sporothrix schenckii.
 9. The method ofclaims 1 or 2 wherein the fluorescent dye binds to DNA of the cells. 10.The method of claims 1 or 2 wherein the fluorescent dye is selected fromthe group consisting of: acridine orange, Hoechst 33258, PicoGreen™,SYTO® 16, SYBR® Green I, Texas Red®, Redmond Red™, Bodipy® Dyes, andOregon Green™.
 11. The method of claims 1 or 2 wherein the fluorescentdye is SYTO®
 16. 12. The method of claim 9 further comprising treatingthe sample with DNase before contacting the sample with the fluorescentdye.
 13. The method of claim 12 further comprising treating the samplewith an agent that affects a cell membrane property of the cells. 14.The method of claim 13 wherein the agent is a detergent.
 15. A kit fordetecting viable cells in a sample, comprising a cell suspensionsolution, a fluorescent dye that can be internalized predominantly byviable cells, and instructions for detecting dye binding to cellularcomponents of viable cells.
 16. The kit of claim 15 wherein the cellsuspension solution comprises a DNase.
 17. The kit of claim 15 whereinthe cell suspension solution comprises an agent that affects a cellmembrane property of the viable cells.
 18. The kit of claim 17 whereinthe agent is a detergent.
 19. The kit of claim 17 wherein thefluorescent dye is selected from the group consisting of: acridineorange, Hoechst 33258, PicoGreen™, SYTO® 16, SYBR® Green I, Texas Red®,Redmond Red™, Bodipy® Dyes, and Oregon Green™.
 20. The kit of claim 19wherein the fluorescent dye is SYTO®
 16. 21. A kit for detecting orquantifying viable cells, comprising: a first container containing afirst solution, means for placement of a sample containing an unknownnumber of viable cells into a solution containing a fluorescent dye thatcan be internalized predominately by the viable cells and bindsselectively to double-stranded DNA or other specific cellularcomponents, whereupon its fluorescence is altered to a measurabledegree, and means for illuminating the mixture of first solution withsaid sample with excitation light and measuring fluorescence emitted bysaid mixture, thereby detecting the presence of viable cells in saidsample.
 22. A kit for detecting or quantifying viable cells, comprising:a first container containing a first solution, means for placement of asample containing an unknown number of viable cells into a firstsolution, means for concentrating the solids and cells from the mixtureof said first solution with said sample and retaining said solids fromthe remainder of said mixture, a second solution containing afluorescent dye that can be internalized predominately by the viablecells and binds selectively to double-stranded DNA or other specificcellular components, whereupon its fluorescence is altered to ameasurable degree, means for mixing said second solution with saidsolids to form a second mixture, and means for illuminating the mixtureof said second solution with said solids with excitation light andmeasuring fluorescence emitted by said mixture, thereby detecting viablecells in said sample.
 23. The kit of claim 21 or 22 wherein thefluorescent dye in said solution is SYTO® 16.